A dUTPase gene (du) deleted ovine lentivirus (OvLV Δdu) mutant, derived from Visna/maedi virus (VMV) molecular clone KV 1772, was constructed. Subsequently, a copy of the optimized green fluorescent protein (egfp) coding region was fused into the viral pol open reading frame (ORF) at the deleted du locus to generate viral mutant, OvLV Δdu-egfp. OvLV Δdu reverse transcriptase (RT) activity and titer of infectious virus in goat synovial membrane (GSM) cell cultures were not affected compared to that of KV1772 and OvLV-85/34 strain (p < 0.05). By contrast, OvLV Δdu-egfp RT activity and virus titer were lower than for KV 1772 and OvLV Δdu (p < 0.05). OvLV-85/34 RT in sheep monocyte-derived macrophages (SMDM) was higher than that of KV 1772, OvLV Δdu and OvLV Δdu-egfp (p < 0.05). The ability to prevent dUTP mis-incorporation into newly synthesized DNA was disrupted in OvLV Δdu and OvLV Δdu-egfp (p < 0.05). Immunoprecipitation demonstrated that GFP is expressed by OvLV Δdu-egfp at a low level. OvLV Δdu-egfp retained egfp after 10 passages in cell culture. OvLV Δdu-egfp was re-isolated in GSM cells from peripheral blood mononuclear (PBMN) cells of three of four OvLV Δdu-egfp-inoculated lambs, but by contrast to the in vitro experiments OvLV Δdu-egfp lost the insert. Priming with OvLV Δdu-egfp did not prevent infection with pathogenic OvLV, but cell-associated viremia in a mock-infected contact control lamb was higher than in OvLV Δdu-egfp-primed lambs. OvLV serum antibody titers increased steadily in OvLV Δdu-egfp-inoculated lambs, but in a lamb from which OvLV Δdu-egfp was not reisolated the antibody titer surpassed the negative/positive cut-off value only after challenge with OvLV-85/34. Because OvLV Δdu-egfp is attenuated for pathogenicity in vitro, replicates in vivo and stimulates an antibody response, subsequent experiments need to address the likelihood of using OvLV Δdu-egfp as an attenuated, live-virus vaccine to protect sheep against OvLV-induced disease when challenged with pathogenic OvLV.
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